Mct-1, a human oncogene

ABSTRACT

A novel gene, designated MCT-1 (for Multiple Copies in T-cell malignancy), is provided. A protein encoded by MCT-1, designated MCT-1, is also provided. Antisense oligonucleotides complementary to or homologous with a portion of MCT-1, substantially purified MCT-1, and methods of determining whether a cell is a tumor cell are also provided. The invention also includes monoclonal and polyclonal antibody preparations which bind with specificity to MCT-1. The invention further includes methods of determining whether a compound or a gene product is a modulator of MCT-1 expression, a method of reducing MCT-1 expression in a cell, and a method of conferring a growth advantage on a cell.

This application is a divisional application of U.S. Pat. No.09/709,131, filed on Nov. 10, 2000, which is in turn a continuation ofinternational patent application PCT/US99/10184, filed on May 10, 1999,which is in turn entitled to priority to U.S. provisional patentapplication 60/085,029, filed on May 11, 1998, the disclosures of eachof which are incorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention is oncogenes and tumorigenesis.

BACKGROUND OF THE INVENTION

Tumorigenesis is a multi-step process wherein a causal relationshipexists between accumulation of genetic abnormalities and aggressiveclinical behavior of tumor cells (Fearon et al., 1990, Cell 61:759-767;Callfano et al., 1996, Cancer Res 56:2488-2492). In many tumors,amplification of critical growth-inducing genes is observed, coupledwith deregulated expression of G1 cyclins and their cognate cdk partners(Lammie et al, 1991, Oncogene 6:439-444; Motokura et al., 1991, Nature350:512-515).

Distinct complexes are formed between cyclins and one or more cognatecyclin dependent kinases (cdks) at different phases of the cell cycle,and activation of the cognate kinases occurs following complexformation. Progression of cells through the late G1 phase of the cellcycle is controlled by G1 cyclins, including D and E type cyclins andtheir cognate cdks (Sherr, 1994, Cell 79:551-555). Phosphorylation ofthe retinoblastoma gene product (Rb) and related gene productsfacilitates entry into S phase. The D type cyclins are known to formcomplexes with either cdk4 or cdk6 (Baldin et al., 1993, Genes Develop.7:812-821).

Expression of G1 cyclins and their cognate cdk partners is oftenderegulated in human tumor cells. Overexpression of cyclin D1 canshorten the G1 interval of the cell cycle, and thereby reduce cell sizeand/or transform cells, both in vitro and in vivo (Jiang et al., 1993,Oncogene 8:3447-3457; Lovec et al., 1994, Oncogene 9:323-326).

Primary cutaneous lymphomas are among the more common presentations ofextra-nodal non-Hodgkins lymphomas (NHLs). NHLs include adult T-cellleukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL). Theetiologic agent of ATLL, HTLV-1, has been known for years. In contrast,the molecular pathogenesis of CTCL, the most frequent type of cutaneouslymphoma, is not well characterized at present. A recent reportdemonstrated rearrangement of the tal-1 and lyt-10 genes in a smallsubset of CTCL cells, but no consistent molecular lesions were detected(Neri et al., 1995, Blood 86:3160-3172).

Development of effective anti-cancer or anti-tumorigenic treatmentswould be facilitated by identification of one or more genes,mal-expression of which is associated with the onset or progression oftumorigenesis. The present invention provides the identity and thecoding sequence of such a gene.

BRIEF SUMMARY OF THE INVENTION

The invention relates to an isolated nucleic acid which binds with highspecificity with a portion of the mRNA-coding region of a human MCT-1gene. In one embodiment, the mRNA-coding region of the gene has thenucleotide sequence SEQ ID NO: 7. For example, the portion may have anucleotide sequence selected from the group consisting of at least about20 or 25 consecutive nucleotide residues of SEQ ID NO: 7 and at leastabout 20 or 25 consecutive nucleotide residues of the sequencecomplementary to SEQ ID NO: 7. In another embodiment, the isolatednucleic acid has a sequence which is either at least about 75%homologous with the portion or at least about 75% complementary to theportion. For example, the isolated nucleic acid may have a sequencewhich is either at least about 95% or 100% homologous with the portionor at least about 95% or 100% complementary to the portion.

The isolated nucleic acid of the invention may comprises one or moremodified internucleoside linkages.

The invention includes a vector comprising the isolated nucleic acid ofthe invention. In the vector, the isolated nucleic acid may be operablylinked with a promoter. For example, a portion comprising nucleotideresidues 258-800 of SEQ ID NO: 7 may be operably linked with a promoter.

The invention also includes a pair of isolated nucleic acids of theinvention wherein one of the pair is complementary to a first portion ofthe mRNA-encoding region and the other of the pair is homologous with asecond portion of the mRNA-encoding region.

The invention further includes an isolated molecular beacon nucleic acidcomprising a first portion and a second portion. The first portion bindswith high specificity with a region of the mRNA-coding region of a humanMCT-1 gene. The second portion anneals with the first portion to alesser degree when the first portion is not annealed with the regionthan when the first portion is annealed with the region. The firstportion has one of a fluorophore-quencher pair associated therewith, andthe second portion has the other of the fluorophore-quencher pairassociated therewith. The molecular beacon nucleic acid of the inventionfluoresces in the presence of the region to a greater degree than in theabsence of the region.

In another aspect, the invention relates to an isolated polypeptidehaving an amino acid sequence which comprises at least about ten orfifteen consecutive amino acid residues of SEQ ID NO: 8. The amino acidsequence of the isolated polypeptide may, of course, be the entirety ofSEQ ID NO: 8. In one embodiment, the polypeptide is substantiallypurified.

The invention also relates to a method of reducing MCT-1 expression in acell. This method comprises providing an isolated nucleic acid whichbinds with high specificity with a portion of the mRNA-coding region ofa human MCT-1 gene to the cell. Expression of MCT-1 in the cell isthereby reduced.

The invention further relates to a method of increasing MCT-1 productionin a cell. This method comprises providing an isolated nucleic acid tothe cell. The isolated nucleic acid comprises a promoter operably linkedwith a portion of the mRNA-coding region of an MCT-1 gene. Production ofMCT-1 in the cell is increased by providing the isolated nucleic acid tothe cell. In one embodiment of this method, the portion comprisesnucleotide residues 258-800 of SEQ ID NO: 7.

The invention still further relates to a method of determining whether atest compound is a modulator of MCT-1 expression. This method comprisesculturing a first cell which overexpresses MCT-1 in the presence of thetest compound and comparing MCT-1 expression in the first cell withMCT-1 expression in a second cell of the same type cultured in theabsence of the test compound. A difference between MCT-1 expression inthe first cell and MCT-1 expression in the second cell is an indicationthat the test compound is a modulator of MCT-1 expression.

The invention also includes a method of determining whether a geneproduct is a modulator of MCT-1 expression. This method comprisesexpressing an isolated nucleic acid encoding the gene product in a firstcell which overexpresses MCT-1 and comparing MCT-1 expression in thefirst cell with MCT-1 expression in a second cell of the same type. Theisolated nucleic acid is not expressed in the second cell. A differencebetween MCT-1 expression in the first cell and MCT-1 expression in thesecond cell is an indication that the gene product is a modulator ofMCT-1 expression.

The invention further includes a method of determining whether a cell isa tumor cell. This method comprises comparing MCT-1 expression in thecell and MCT-1 expression in a non-tumor cell. A difference betweenMCT-1 expression in the cell and MCT-1 expression in the non-tumor cellis an indication that the cell is a tumor cell.

In another aspect, the invention relates to a method of determiningwhether a cell is a tumor cell. This method comprises comparing MCT-1copy number in the cell and MCT-1 copy number in a non-tumor cell. Adifference between MCT-1 copy number in the cell and MCT-1 copy numberin the non-tumor cell is an indication that the cell is a tumor cell.

The invention also includes a method of conferring a growth advantage ona cell. This method comprises providing an isolated nucleic acid to thecell. The isolated nucleic acid comprises a promoter operably linked toa portion of the mRNA-coding region of an MCT-1 gene. By providing thisisolated nucleic acid to the cell, a growth advantage is conferred onthe cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, comprising FIGS. 1A and 1B is a pair of sequences. FIG. 1A is anucleotide sequence (SEQ ID NO: 1) of a 944 nucleotide residue humancDNA molecule described in Example 1, which was originally thought toencode MCT-1. It is now known that the cytosine residue at position 599is not present in the sequence, as indicated in the corrected sequencelisted in FIG. 5A. FIG. 1B is the deduced amino acid sequence (SEQ IDNO: 2) of the protein encoded by the nucleotide sequence listed in FIG.1A. Single-letter codes are used to identify amino acid residues. It isnow known that the amino acid sequence of residues 115+ of MCT-1 differsfrom that listed here, as indicated in the corrected sequence listed inFIG. 5B.

FIG. 2 is a diagram which depicts various polynucleotides. The top lineis a scale which indicates polynucleotide length, measured in nucleotideresidues. The second line depicts MCT-1 mRNA; the region of the moleculecorresponding to the coding region of MCT-1 is indicated by a thick bar.The third line depicts a 3′-RACE (rapid amplification of cDNA ends)product prepared using MCT-1 mRNA. The fourth line depicts a 5′-RACEproduct prepared using MCT-1 mRNA. The fifth through fourteenth linesrepresent individual expressed sequence tags (ESTs) which exhibitedhomology with MCT-1.

FIG. 3 is a listing of the amino acid sequence of MCT-1 (SEQ ID NO: 2),wherein putative post-translational modification sites are indicated. Aputative glycosylation site, a putative Tyr phosphorylation site, twoputative PKC phosphorylation sites, and a putative CK2 phosphorylationsite are indicated.

FIG. 4 is a comparison of a portion (SEQ ID NO: 9) of the amino acidsequence of MCT-1 with a similar portion (SEQ ID NO: 10) of the aminoacid sequence of Cyclin H. A solid bar between adjacent amino acidresidues indicates identity; double dots between adjacent amino acidresidues indicates conservative replacement; single dots betweenadjacent amino acid residues indicates amino acid residues which arestructurally similar.

FIG. 5, comprising FIGS. 5A and 5B, is a pair of sequence listings. Thenucleotide sequence (SEQ ID NO: 7) of the cDNA encoding MCT-1 is listedin FIG. 5A. The amino acid sequence (SEQ ID NO: 8) of MCT-1 is listed inFIG. 5B. The underlined sequence is the polyadenylation signal.

DETAILED DESCRIPTION

The invention relates to the discovery of a gene, herein designatedMCT-1 (for Multiple Copies in T-cell malignancy), which is overexpressedin certain tumor cells. For example, MCT-1 is overexpressed in T-celltumor cells, such as cells obtained from a patient afflicted withcutaneous T-cell leukemia (CTCL; i.e. the Hut 78 cell line). Genomicanalysis of such cells indicated that the MCT-1 gene is present in anincreased copy number in tumor cell lines established from a patient.The MCT-1 gene comprises an open reading frame (ORF) which encodes a181-amino acid residue polypeptide, herein designated MCT-1. A cDNAmolecule comprising the ORF of MCT-1 has been isolated (SEQ ID NO: 7;listed in FIG. 5A). MCT-1 has the amino acid sequence SEQ ID NO: 8, andis listed in FIG. 5B.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein and mean any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil).

A nucleic acid has a “modified internucleoside linkage” if at least onephosphodiester bond in the nucleic acid is replaced by an alternativechemical linkage such as, for example, a phosphoramidate linkage.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked with the coding region of a geneis able to promote transcription of the coding region.

As used herein, the term “promoter” means a DNA sequence which isrequired for expression of a gene operably linked to the promoter. Insome instances, the promoter may be the core promoter sequence and inother instances, the promoter may also include an enhancer sequence andother regulatory elements which are required for expression of the genein, for example, an inducible, suppressible, or tissue-specific manner.

A “molecular beacon” nucleic acid is a nucleic acid comprising a pair ofcomplementary regions and having a fluorophore and a fluorescentquencher associated therewith. The fluorophore and quencher areassociated with different portions of the nucleic acid in such anorientation that when the complementary regions are annealed with oneanother, fluorescence of the fluorophore is quenched by the quencher.When the complementary regions of the nucleic acid are not annealed withone another, fluorescence of the fluorophore is quenched to a lesserdegree. Molecular beacon nucleic acids are described, for example, inU.S. Pat. No. 5,876,930.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.

An “mRNA-coding region” of a gene consists of the nucleotide residues ofthe coding strand of the gene and the nucleotide residues of thenon-coding strand of the gene which are homologous with or complementaryto, respectively, an mRNA molecule which is produced by transcription ofthe gene. It is understood that, owing to mRNA processing which occursin certain instances in eukaryotic cells, the mRNA-coding region of agene may comprise a single region or a plurality of regions separatedfrom one another in the gene as it occurs in the genome. Where themRNA-coding region of a gene comprises separate regions in a genome,“mRNA-coding region” refers both individually and collectively to eachof these regions.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′-ATTGCC-5′ and 3′-TATGGC-5′ share 50%homology. When every subunit position in the two molecules is occupiedby the same monomeric subunit, the two molecules are said to be“completely” homologous.

“Complementary” refers to the broad concept of subunit sequencecomplementarity between regions of two nucleic acid strands or betweentwo regions of the same nucleic acid strand. It is known that an adenineresidue of a first nucleic acid region is capable of forming specifichydrogen bonds (“base pairing”) with a residue of a second nucleic acidregion which is antiparallel to the first region if the residue isthymine or uracil. Similarly, it is known that a cytosine residue of afirst nucleic acid strand is capable of base pairing with a residue of asecond nucleic acid strand which is antiparallel to the first strand ifthe residue is guanine. A first region of a nucleic acid iscomplementary to a second region of the same or a different nucleic acidif, when the two regions are arranged in an antiparallel fashion, atleast one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion, in which instance thetwo portions are described as being “completely” complementary.

A first oligonucleotide anneals with a second oligonucleotide “with highstringency” if the two oligonucleotides anneal under high stringencyhybridization conditions.

By “high stringency hybridization conditions” is meant thoseoligonucleotide hybridizing conditions that (1) employ low ionicstrength and high temperature for washing, for example, 0.015 molarNaCl, 1.5 millimolar sodium citrate, and 0.1% (w/v) sodium dodecylsulfate (SDS) at 50° C.; (2) employ during hybridization a denaturingagent such as formamide, for example, 50% (v/v) formamide, 0.1% (w/v)bovine serum albumin, 0.1% (w/v) Ficoll, 0.1% (w/v)polyvinylpyrrolidone, and 50 millimolar sodium phosphate buffer at pH6.5 with 750 millimolar NaCl, 75 millimolar sodium citrate at 42° C.; or(3) employ 50% (v/v) formamide, 5×SSC (0.75 molar NaCl, 75 millimolarsodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 micrograms per milliliter), 0.1% (w/v) SDS, and 10% (w/v) dextransulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% (w/v) SDS.Under stringent hybridization conditions, only highly complementarynucleic acids hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides.

A “portion” and a “region” of a polynucleotide are used interchangeablyto mean at least at least about twenty sequential nucleotide residues ofthe polynucleotide. It is recognized that a portion of a polynucleotidemay include every nucleotide residue of the polynucleotide.

As used herein, an “amplified genomic sequence” is a sequence ofnucleotide residues in the genome of a mammal such as a human which ispresent in the genome in a greater number of copies than the number ofcopies normally present in the genome.

The term “substantially purified” describes a compound, e.g., a proteinor polypeptide which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

“Malexpression” of a gene means expression of a gene in a cell of apatient afflicted with a disease or disorder, wherein the level ofexpression (including non-expression), the portion of the geneexpressed, or the timing of the expression of the gene with regard tothe cell cycle, differs from expression of the same gene in a cell of apatient not afflicted with the disease or disorder. It is understoodthat malexpression may cause or contribute to the disease or disorder,be a symptom of the disease or disorder, or both.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient(s) may be combinedand which, following the combination, can be used to administer theactive ingredient(s) to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient(s) which iscompatible with any other ingredients of the pharmaceutical compositionand which is not deleterious to the subject to which the composition isto be administered.

The Isolated Nucleic Acid of the Invention

The invention relates to an isolated nucleic acid which binds with highspecificity with a portion of the mRNA-coding region of a human MCT-1gene. This region is listed in FIG. 5A (SEQ ID NO: 7). The isolatednucleic acid may be homologous with or complementary to a portion of SEQID NO: 7, and includes nucleotide residues 258-800 of SEQ ID NO: 7,which encode the amino acid sequence of MCT-1 (SEQ ID NO: 8). Theportion may be homologous with or complementary to the entiremRNA-coding region, or at least about twenty, twenty-five, thirty,fifty, or more nucleotide residues thereof. It is understood that theisolated nucleic acid of the invention need not be completely homologouswith or completely complementary to the mRNA-coding region in order forit to bind with high specificity therewith. Instead, the isolatednucleic acid need only be mostly complementary or homologous. Forexample, the isolated nucleic acid may be 75%, 90%, 95%, or, preferably,completely complementary to or homologous with the mRNA-coding region.

In an important embodiment of the present invention, the isolatednucleic acid has a sequence which is homologous with nucleotide residues258-800 of SEQ ID NO: 7. This portion of the mRNA-coding region encodesthe amino acid sequence of MCT-1 protein. By operably linking thisportion, or at least most residues thereof (e.g. the nucleotide residuesencoding amino acid residues 8-65 of MCT-1) with a transcriptionalpromoter, the resulting nucleic acid may be used to generate MCT-1protein, either by providing the nucleic acid to an organism capable ofexpressing it or by using an in vitro transcription/translation mixture.Because this portion does not include the terminator codon (i.e.nucleotide residues 801-803 of SEQ ID NO: 7), a termination sequenceshould be operably associated with the portion if the portion is to beexpressed. Of course, it is understood that additional aminoacid-specifying codons may be inserted between the portion and either orboth of the promoter region or the termination sequence, whereby afusion protein comprising at least a portion of the amino acid sequenceof MCT-1 is generated upon expression of the nucleic acid. The fusionprotein may, for example, comprise a portion of a hemagglutinin (asdescribed herein in Example 2) or a hexa-histidine polypeptide sequencefor facilitating purification of the expressed protein by metal-(e.g.nickel-)affinity chromatography.

Given the nucleotide sequence of the mRNA-coding region of MCT-1provided herein, one skilled in the art can generate a polynucleotidecapable of annealing with either the coding or the non-coding strand ofMCT-1 or with RNA, such as mRNA, transcribed therefrom. Sucholigonucleotides are useful for binding to mRNA and single-stranded DNAto modulate transcription and translation thereof, to facilitatespecific detection thereof, or to amplify a sequence using a PCR method,for example.

For example, an antisense oligonucleotide capable of annealing with thecoding strand of the mRNA-coding region of MCT-1 (and therefore alsocapable of annealing with an mRNA molecule generated by transcribingMCT-1) is made by generating an oligonucleotide having a nucleotidesequence complementary to a portion of SEQ ID NO: 7. An antisenseoligonucleotide capable of annealing with the non-coding strand of themRNA-coding region of MCT-1 is made by generating an oligonucleotidehaving a nucleotide sequence homologous with a portion of SEQ ID NO: 7.Preferably, the antisense oligonucleotide is either complementary to orhomologous with at least about twenty sequential nucleotide residues ofSEQ ID NO: 7, and preferably to or with about twenty-five, thirty,fifty, or more sequential nucleotide residues. As is understood in theart, regions of homology between two polynucleotides may be interruptedby one or more non-homologous base pairs. Thus, the antisenseoligonucleotide may comprise one or more nucleotide residues which arenot homologous or complementary to the portion of SEQ ID NO: 7. Anantisense oligonucleotide complementary to a portion of SEQ ID NO: 7 isuseful for inhibiting translation of an mRNA molecule generated bytranscribing MCT-1. An antisense oligonucleotide homologous with aportion of SEQ ID NO: 7 is useful for inhibiting transcription of MCT-1.

In another important embodiment, the isolated nucleic acids of theinvention are supplied in pairs which are useful for amplification ofall or part of the sequence of the human MCT-1 gene. Design and use ofsuch primer pairs for amplification of nucleic acids are well known inthe art and are fully enabled by the listing herein of SEQ ID NOs: 1 and7. Such primer pairs will generally comprise a first isolated nucleicacid which is complementary to a first portion of SEQ ID NO: 7 and asecond isolated nucleic acid which is complementary to a second portionof SEQ ID NO: 7.

It is understood that the sole sequence difference between SEQ ID NO: 7and SEQ ID NO: 1 is the presence of an additional cytosine residue inSEQ ID NO: 1 at position 599 (i.e. between residues 598 and 599 of SEQID NO: 7). Thus, SEQ ID NO: 1 may be used in place of SEQ ID NO: 7herein, except that use of SEQ ID NO: 7 is preferred when the identityof the residue at position 599 is critical.

The isolated nucleic acid of the invention may be delivered to a cellusing a vector. The vector may comprise the isolated nucleic acidoperably linked with one or more of a promoter sequence, atranscriptional or translational regulatory sequence, amembrane-directing or “signal” sequence, and a terminator sequence, orit may not be operably linked with any of these. Thus, the isolatednucleic acid may be merely delivered to a cell by the vector, bedelivered to the cell in a form in which it is transcribed in the cell,be delivered in a form in which it is transcribed and translated in thecell, or be delivered in a form in which it is transcribed, translated,and directed to a particular location relative to the cell (e.g. to thenucleus or to the exterior of the cell). All of these sequences andtheir use are well known in the art. The vector may, for example, be aviral vector or a non-viral vector such as a plasmid.

Numerous modifications of oligonucleotides are known in the art, andthese modifications, as applied to the isolated nucleic acid of theinvention, are also included in the invention. For example,oligonucleotides comprising altered sugar moieties, non-naturalinter-sugar linkages, phosphorothioate moieties, methyl phosphonatemoieties, short chain alkyl moieties, cycloalkyl moieties, and the likeare known.

It is not intended that the present invention be limited by the natureof the nucleic acid employed. The isolated nucleic acid of the inventionmay be native or synthesized nucleic acid. The isolated nucleic acid maybe obtained from a viral, bacterial, animal, plant, or synthetic source.The nucleic acid may be DNA or RNA and may exist in a double-stranded,single-stranded or partially double-stranded form. Furthermore, thenucleic acid may be found as part of a virus or other macromolecule.See, e.g., Fasbender et al., 1996, J. Biol. Chem. 272:6479-89(polylysine condensation of DNA in the form of adenovirus).

Nucleic acids useful in the present invention include, by way of exampleand not limitation, oligonucleotides and polynucleotides such asantisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viralfragments including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA;plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structuralforms of DNA including single-stranded DNA, double stranded DNA,supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like. Thenucleic acids may be prepared by any conventional means typically usedto prepare nucleic acids in large quantity. For example, DNAs and RNAsmay be chemically synthesized using commercially available reagents andsynthesizers by methods that are well-known in the art (see, e.g., Gait,1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press,Oxford, England)). RNAs may be produce in high yield via in vitrotranscription using plasmids such as SP65 (Promega Corporation, Madison,Wis.).

In some circumstances, as where increased nuclease stability is desired,isolated nucleic acids having modified internucleoside linkages may bepreferred. Isolated nucleic acids containing modified internucleosidelinkages may also be synthesized using reagents and methods that arewell known in the art. For example, methods for synthesizing nucleicacids containing phosphonate phosphorothioate, phosphorodithioate,phosphoramidate methoxyethyl phosphoramidate, formacetal,thioformacetal, diisopropylsilyl, acetamidate, carbamate,dimethylene-sulfide (—CH₂—S—CH₂—), dimethylene-sulfoxide (—CH₂—SO—CH₂—),dimethylene-sulfone (—CH₂—SO₂—CH₂—), 2′-O-alkyl, and 2′-deoxy-2′-fluorophosphorothioate internucleoside linkages are well known in the art(e.g. Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al.,1990, Tetrahedron Lett. 31:335).

The isolated nucleic acid of the invention may be purified by anysuitable means, as are well known in the art. For example, the nucleicacids can be purified by reverse phase or ion exchange HPLC, sizeexclusion chromatography or gel electrophoresis. Of course, the skilledartisan will recognize that the method of purification will depend inpart on the size and type of the nucleic acid to be purified and on thecharacteristics of any molecules, structure, or organisms with which itmay be associated. It is furthermore contemplated that the nucleic acidmay comprise nucleotide residues other than the five naturally occurringbases, adenine, guanine, thymine, cytosine, and uracil.

Also contemplated is a manufacture comprising a plurality of isolatednucleic acids (i.e. probes) of the invention fixed in an ordered arrayon a surface. Each of the plurality of probes anneals with highstringency with a portion of the human MCT-1 gene. By including probeswhich differ by a single nucleotide residue within the correspondingportion of the MCT-1 gene, nucleic acids which comprise differentnucleotide residues at that position within the MCT-1 gene may bedifferentiated. Thus, using methods well known in the art, missense anddeletion mutations in the MCT-1 sequence may be detected. Furthermore,by incorporating into the array probes which bind with high affinitywith sequential portions of the wild type MCT-1 gene, wherein eachsequential portion includes one nucleotide residue not included withinthe previous sequential portion, the nucleotide sequence of all, or anyportion, of the MCT-1 gene may be determined. Preferably, the wild typehuman MCT-1 cDNA sequence which is used is SEQ ID NO: 7). Manufacturesof this type are analogous to the GeneChip™ devices manufactured byAffymetrix, Inc. (Santa Clara, Calif.), which comprise pluralities ofprimers which bind with high stringency to, for example, portions of thehuman p53 gene or to portions of the HIV-1 protease or reversetranscriptase genes. Methods for making and using such manufactures havebeen described elsewhere, and need only be modified by the skilledartisan to include the MCT-1 gene sequences described in the presentdisclosure (Wallraff et al., February 1997, Chemtech 22-23; Lockhart etal., 1996, Nature Biotechnol. 14:1675-1680; Pease et al., 1994, Proc.Natl. Acad. Sci. USA 91:5022-5026; Fodor et al., 1993, Nature364:555-556).

The Isolated Polypeptide of the Invention

The invention further relates to an isolated polypeptide which ishomologous with at least a portion of MCT-1. The isolated polypeptide ofthe invention is preferably homologous with at least about ten, fifteen,twenty, or more amino acid residues of SEQ ID NO: 8, which is listed inFIG. 5B. It is understood that SEQ ID NOs: 2 and 8 are identical atamino acid residues 1 to 114. Therefore, these two sequences may be usedinterchangeably, except that use of SEQ ID NO: 8 is preferred when theidentity of one or more of amino acid residues 115 to 181 is critical.In one embodiment, the isolated polypeptide of the invention comprisesamino acid residues 8 to 65 of SEQ ID NO: 8 (or SEQ ID NO: 2). Thisportion is highly similar to a region of cyclin H protein which has beenimplicated in protein-protein interactions. Thus, it is recognized thatthis region of MCT-1 is likely to be at least a significant portion ofMCT-1 which interacts with the proteins by means of which MCT-1 exertsits biological effect.

The isolated polypeptide of the invention may have a sequence whichcomprises all or part of SEQ ID NO: 8. For example, the isolatedpolypeptide of the invention may be MCT-1 protein, preferably in asubstantially purified form. In addition, the sequence of the isolatedpolypeptide of the invention may further comprise additional amino acidresidues (i.e. it may be a fusion protein) or comprise amino acidsubstitutions, particularly in the random coil portions of MCT-1.

As described herein, MCT-1 has been purified by expressing a GST-MCT-1fusion protein in an Escherichia coli vector and isolating the fusionprotein from the vector using known methods. Monoclonal or polyclonalantibodies which bind with specificity to MCT-1 may be generated usingknown methods and are included in the invention. Antibodies which bindwith specificity to MCT-1 are useful for detecting the presence of MCT-1in a cell, and thus can be used to determine whether a cell is a tumorcell. To determine whether a cell is a tumor cell, an antibody whichbinds with specificity to MCT-1 is used to detect the presence of MCT-1in an extract prepared using the cell, using any immunoblotting,immunosorption, or immunoprecipitation technique. The presence of MCT-1in the cell is an indication that the cell is a tumor cell.

The locations of regions of random coil in the amino acid sequence ofMCT-1 may be predicted using standard sequence analysis algorithms (e.g.Gamier-Robson analysis). In random coil regions of proteins, the aminoacid sequence is relatively unimportant with regard to the biologicalactivity of the protein. Thus, individual amino acid residues in regionsof random coil in SEQ ID NO: 8 may be substituted with substantially anyamino acid residue. Other amino acid residues in SEQ ID NO: 8 shouldonly be substituted with conservative amino acid residues. Conservativeamino acid residue substitutions are listed on rows of the followingtable.

TABLE glycine, alanine valine, isoleucine, leucine aspartic acid,glutamic acid asparagine, glutamine serine, threonine lysine, argininephenylalanine, tyrosine

It will be appreciated, of course, that the peptides may incorporateamino acid residues which are modified without affecting biologicalactivity. For example, the termini may be derivatized to includeblocking groups, i.e. chemical substituents suitable to protect and/orstabilize the N- and C-termini from “undesirable degradation”, a termmeant to encompass any type of enzymatic, chemical or biochemicalbreakdown of the compound at its termini which is likely to affect thefunction of the compound as an anti-inflammatory agent, i.e. sequentialdegradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor non-branched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectinganti-tumorigenic activity and these include, but are not limited to,substitution of one or more of the amino acids in the natural L-isomericform with amino acids in the D-isomeric form. Thus, the peptide mayinclude one or more D-amino acid resides, or may comprise amino acidswhich are all in the D-form. Retro-inverso forms of peptides inaccordance with the present invention are also contemplated, forexample, inverted peptides in which all amino acids are substituted withD-amino acid forms.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, an isolated polypeptide of the inventionmay be treated with an inorganic acid such as hydrochloric, hydrobromic,sulfuric, nitric, phosphoric, and the like, or an organic acid such asan acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic,succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylicand the like, to provide a water soluble salt of the polypeptide issuitable for use in the methods described herein.

Pharmaceutical Compositions

The invention encompasses the preparation and use of medicaments andpharmaceutical compositions comprising either or both of an isolatednucleic acid of the invention and an isolated polypeptide of theinvention as an active ingredient. Such a pharmaceutical composition mayconsist of the active ingredient(s) alone, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient(s) and one or more pharmaceuticallyacceptable carriers, one or more additional ingredients, or somecombination of these. Administration of one of these pharmaceuticalcompositions to a subject is useful for preventing or inhibitingtumorigenesis in the subject or for treating a pre-existing tumor, asdescribed elsewhere in the present disclosure. The active ingredient(s)may be present in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient(s) into association with a carrier or oneor more other accessory ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desired single- ormulti-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient(s),and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient(s). The amount of the active ingredient(s) isgenerally equal to the dosage of the active ingredient(s) which would beadministered to a subject or a convenient fraction of such a dosage suchas, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient(s), the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient(s).

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient(s). Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient(s) may, for example, be madeby compressing or molding the active ingredient(s), optionally with oneor more additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient(s) in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient(s), apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycolate. Known surface active agents include,but are not limited to, sodium lauryl sulphate. Known diluents include,but are not limited to, calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient(s). By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient(s) may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient(s), and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient(s) may be madeusing a physiologically degradable composition, such as gelatin. Suchsoft capsules comprise the active ingredient(s), which may be mixed withwater or an oil medium such as peanut oil, liquid paraffin, or oliveoil.

Oral compositions may be made, using known technology, whichspecifically release orally-administered agents in the small or largeintestines of a human patient. For example, formulations for delivery tothe gastrointestinal system, including the colon, include enteric coatedsystems, based, e.g., on methacrylate copolymers such aspoly(methacrylic acid, methyl methacrylate), which are only soluble atpH 6 and above, so that the polymer only begins to dissolve on entryinto the small intestine. The site where such polymer formulationsdisintegrate is dependent on the rate of intestinal transit and theamount of polymer present. For example, a relatively thick polymercoating is used for delivery to the proximal colon (Hardy et al., 1987Aliment. Pharmacol. Therap. 1:273-280). Polymers capable of providingsite-specific colonic delivery can also be used, wherein the polymerrelies on the bacterial flora of the large bowel to provide enzymaticdegradation of the polymer coat and hence release of the drug. Forexample, azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friend etal., 1984, J. Med. Chem. 27:261-268) and a variety of naturallyavailable and modified polysaccharides (PCT GB 89/00581) may be used insuch formulations.

Pulsed release technology such as that described in U.S. Pat. No.4,777,049 may also be used to administer the active agent to a specificlocation within the gastrointestinal tract. Such systems permit drugdelivery at a predetermined time and can be used to deliver the activeagent, optionally together with other additives that my alter the localmicroenvironment to promote agent stability and uptake, directly to thecolon, without relying on external conditions other than the presence ofwater to provide in vivo release.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient(s) in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient(s) in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient(s) is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient(s) in the solvent. Aqueous solvents include,for example, water and isotonic saline. Oily solvents include, forexample, almond oil, oily esters, ethyl alcohol, vegetable oils such asarachis, olive, sesame, or coconut oil, fractionated vegetable oils, andmineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the activeingredient(s) with a non-irritating pharmaceutically acceptableexcipient which is solid at ordinary room temperature (i.e. about 20°C.) and which is liquid at the rectal temperature of the subject (i.e.about 37° C. in a healthy human). Suitable pharmaceutically acceptableexcipients include, but are not limited to, cocoa butter, polyethyleneglycols, and various glycerides. Suppository formulations may furthercomprise various additional ingredients including, but not limited to,antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient(s) with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient(s) with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intravenous, intraarterial, intramuscular, orintrasternal injection and intravenous, intraarterial, or kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient(s) combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules, in multi-dose containerscontaining a preservative, or in single-use devices for auto-injectionor injection by a medical practitioner. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and implantablesustained-release or biodegradable formulations. Such formulations mayfurther comprise one or more additional ingredients including, but notlimited to, suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the activeingredient(s) is provided in dry (i.e. powder or granular) form forreconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient(s),additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient(s) in microcrystalline form, in a liposomal preparation, oras a component of a biodegradable polymer systems. Compositions forsustained release or implantation may comprise pharmaceuticallyacceptable polymeric or hydrophobic materials such as an emulsion, anion exchange resin, a sparingly soluble polymer, or a sparingly solublesalt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient(s), although theconcentration of the active ingredient(s) may be as high as thesolubility limit of the active ingredient(s) in the solvent.Formulations for topical administration may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient(s) and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient(s) dissolvedor suspended in a low-boiling propellant in a sealed container.Preferably, such powders comprise particles wherein at least 98% of theparticles by weight have a diameter greater than 0.5 nanometers and atleast 95% of the particles by number have a diameter less than 7nanometers. More preferably, at least 95% of the particles by weighthave a diameter greater than 1 nanometer and at least 90% of theparticles by number have a diameter less than 6 nanometers. Dry powdercompositions preferably include a solid fine powder diluent such assugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient(s) may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient(s)).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient(s) in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient(s),and may conveniently be administered using any nebulization oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration preferably have anaverage diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient(s) and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient(s), and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient(s), the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient(s).Such powdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient(s) in an aqueous or oily liquid carrier. Such drops mayfurther comprise buffering agents, salts, or one or more other of theadditional ingredients described herein. Otherophthalmalmically-administrable formulations which are useful includethose which comprise the active ingredient(s) in microcrystalline formor in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

A pharmaceutical composition of the invention may be administered todeliver a dose of between 1 ng/kg/day and 100 mg/kg/day to a subject.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe an effective amount of the compoundto inhibit or treat a tumor in the subject. In so proceeding, thephysician or veterinarian may, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. It is further understood, however, that thespecific dose level for any particular subject will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, any drug combination, and the stage of progression ofthe tumor being treated or inhibited.

Another aspect of the invention relates to a kit comprising apharmaceutical composition of the invention and an instructionalmaterial. As used herein, an “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionwhich is used to communicate the usefulness of the pharmaceuticalcomposition of the invention for inhibiting or treating a tumor in asubject. The instructional material may also, for example, describe anappropriate dose of the pharmaceutical composition of the invention. Theinstructional material of the kit of the invention may, for example, beaffixed to a container which contains a pharmaceutical composition ofthe invention or be shipped together with a container which contains thepharmaceutical composition. Alternatively, the instructional materialmay be shipped separately from the container with the intention that theinstructional material and the pharmaceutical composition be usedcooperatively by the recipient.

The invention also includes a kit comprising a pharmaceuticalcomposition of the invention and a delivery device for delivering thecomposition to a subject. By way of example, the delivery device may bea squeezable spray bottle, a metered-dose spray bottle, an aerosol spraydevice, an atomizer, a dry powder delivery device, a self-propellingsolvent/powder-dispensing device, a syringe, a needle, a tampon, or adosage measuring container. The kit may further comprise aninstructional material as described herein.

The Methods of the Invention

The invention also relates to a method of reducing MCT-1 expression in acell. The method comprises providing an isolated nucleic acid of theinvention to the cell. Because the isolated nucleic acid iscomplementary to or homologous with at least about twenty consecutivenucleotides of the mRNA-coding region of an MCT-1 gene to the cell, thenucleic acid anneals with DNA or mRNA in the cell and reduces expressionof MCT-1 in the cell. In one embodiment, the isolated nucleic acid iscomplementary to at least about twenty, and preferably at least abouttwenty-five, thirty, fifty, or more nucleotide residues of SEQ ID NO: 7,so that the isolated nucleic acid binds with high specificity to mRNAgenerated by transcription of the genome of the cell and therebyprevents translation of the mRNA, blocking MCT-1 synthesis. The isolatednucleic acid may, for example be complementary to a portion of themRNA-coding region near or including the translational start site (i.e.nucleotide residues 258-260 of SEQ ID NO: 7). By way of example, theisolated nucleic acid may have a sequence which is 75%, 90%, 95%, orcompletely complementary to nucleotide residues 240-270 of SEQ ID NO: 7.

The invention also relates to a method of increasing MCT-1 production ina cell. This method comprises providing to the cell an isolated nucleicacid comprising a promoter operably linked with a portion of themRNA-coding region of a human MCT-1 gene. The region may, for example,include the entire coding sequence of MCT-1 (i.e. nucleotide residues258-800 of SEQ ID NO: 7), or it may include additional (i.e. a fusionprotein) or fewer amino acid residues (i.e. a fragment of MCT-1 such asone including residues 8-65). Of course, the portion of the mRNA-codingregion may have amino acid substitutions, as described elsewhere herein.Production of MCT-1 in the cell is increased by expression of the codingsequence of MCT-1 of the isolated nucleic acid. The promoter which isoperably linked may be an inducible, suppressible, tissue-specific, orconstitutive promoter, each of which is well known in the art.

The invention further relates to a method of determining whether acompound is a modulator of MCT-1 expression. This method of theinvention comprises providing a first cell which overexpresses MCT-1,culturing the first cell in the presence of the compound, and comparingMCT-1 expression in the first cell with MCT-1 expression in a secondcell of the same type cultured in the absence of the compound. Adifference between MCT-1 expression in the first cell and MCT-1expression in the second cell is an indication that the compound is amodulator of MCT-1 expression. The first cell and the second cell mayeach be, for example, a leukocyte which has been obtained from a patientafflicted with CTCL and which overexpresses MCT-1.

Expression of MCT-1 in a first cell and in a second cell may be detectedand compared using known methods, such as those described herein in theExample. It is understood that this method may also be practiced using afirst cell which does not normally express MCT-1 or one which normallyexpresses MCT-1 at a relatively constant level. When a second cell ofthe same type is cultured in the presence of a test compound, comparingexpression of MCT-1 in the first and second cells will indicate whetherthe test compound modulates expression of MCT-1. For example, if thefirst and second cells do not normally express MCT-1, but the secondcell expresses MCT-1 in the presence of a test compound, then this is anindication that the test compound is an inducer of MCT-1 expression. Thetest compound may therefore be considered a potential carcinogen.

The invention also includes a method of determining whether a geneproduct is a modulator of MCT-1 expression. This method comprisesproviding a first cell which overexpresses MCT-1, expressing an isolatednucleic acid encoding the gene product in the first cell, and comparingMCT-1 expression in the first cell with MCT-1 expression in a secondcell of the same type, wherein the isolated nucleic acid is notexpressed in the second cell. A difference between MCT-1 expression inthe first cell and MCT-1 expression in the second cell is an indicationthat the gene product is a modulator of MCT-1 expression. It isunderstood that this method may also be practiced using a first cellwhich does not normally express MCT-1 or one which normally expressesMCT-1 at a relatively constant level.

The invention further includes a method of determining whether a cell isa tumor cell. This method of the invention comprises comparing MCT-1expression in the cell and MCT-1 expression in a non-tumor cell,preferably of the same type (e.g. by comparing MCT-1 expression in aT-cell suspected of being cancerous and in a T-cell not suspected ofbeing cancerous). A difference between MCT-1 expression in the cell andMCT-1 expression in the non-tumor cell is an indication that the cell isa tumor cell. The method can be used, for example, to determine whetherthe cell is a cutaneous T-cell lymphoma cell. The non-tumor cell may,for example, be a normal human lymphocyte.

The invention includes another method of determining whether a cell is atumor cell. This alternate method comprises comparing MCT-1 copy numberin the cell and MCT-1 copy number in a non-tumor cell. A differencebetween MCT-1 copy number in the cell and MCT-1 copy number in thenon-tumor cell is an indication that the cell is a tumor cell. Themethod can be used, for example, to determine whether the cell is acutaneous T-cell lymphoma cell. The non-tumor cell may, for example, bea normal human lymphocyte. The copy number of MCT-1 in a first cell andin a second cell may be detected and compared using known methods, suchas those described herein in the Example.

The invention also relates to a method of conferring a growth advantageon a cell. This method comprises providing the cell with an isolatednucleic acid comprising a promoter operably linked to a portion of themRNA-coding region of an MCT-1 gene, the region including the codingsequence of MCT-1 (i.e. nucleotide residues 258-623 of SEQ ID NO: 1). Byproviding this isolated nucleic acid to the cell, MCT-1 expression isenhanced, as described in the Example, and a growth advantage isconferred upon the cells, relative to cells not provided the isolatednucleic acid.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLE 1 Molecular Cloning and Characterization of a Novel Gene, MCT-1,which is Amplified in a Cutaneous T-Cell Lymphoma Cell Line

The experiments presented in this Example demonstrate thatoverexpression of MCT-1 protein in NIH 3T3 fibroblasts shortens the G1phase of the cell cycle and promotes anchorage independent growth.

Genetic abnormalities of malignant T-cells associated with cutaneousT-cell leukemia (CTCL) were identified using arbitrarilyprimed-polymerase chain reaction (AP-PCR) assay. AP-PCR has been used byothers to detect sub-microscopic genetic alterations which areassociated with tumorigenesis, as described (Welsh et al., 1990, Nucl.Acids Res. 18:7213-7218; Peinado et al., 1992, Proc. Natl. Acad. Sci.USA 89:10065-10069; Kohno et al., 1993, Oncogene 7:103-108).

A panel of T-cell leukemia and lymphoma cell lines was screened foramplified genomic sequences. An amplified genomic sequence wasidentified in a cell line designated HUT 78. This genomic sequence wascloned and sequenced, and the coding sequence encoded by the genomicsequence was determined to have no significant homology to any knowngene sequence in the BLAST genome data base, as determined using theBasic Local Alignment Search Tool. It was therefore concluded that theamplified genomic sequence comprised at least part of a novel gene.

The novel gene was designated MCT-1 (multiple copies in T-cellmalignancies). The amplified genomic sequence encoded a polypeptide of181 amino acids (SEQ ID NO: 8), designated MCT-1. The MotifFindercomputer program was used to identify three putative post-translationalmodification sites in MCT-1, as indicated in FIG. 3. PCR primers weredesigned using the MacVector program, and these primers were used togenerate a 457 nucleotide residue amplification product when genomic DNAwas subjected to PCR. The primers which were used were designated HuT78F, which had the nucleotide sequence 5′-CATTGGAGAC CGCTACACAC AGGAC-3′(SEQ ID NO: 3), and HuT 78R, which had the nucleotide sequence5′-CTGTCAAAAT AGTCCGATGC CACG-3′ (SEQ ID NO: 4). The same primers werealso useful for amplifying cDNA in an RT-PCR assay, confirming that theamplified band obtained during the AP-PCR assay was an exon.

Northern blot analysis revealed ubiquitous low-level expression of a 943nucleotide residue MCT-1 message in normal human tissues. The fulllength cDNA sequence corresponding to the MCT-1 message (hereinafter,“the MCT-1 cDNA”) was determined using a rapid amplification of cDNAends (RACE) method, as described (Frohman, 1993, Meth. Enzymol.218:340-356). The nucleotide sequence (SEQ ID NO: 7) of the MCT-1 cDNAis listed in FIG. 5A, and the amino acid sequence (SEQ ID NO: 8) of the181-amino acid residue polypeptide encoded thereby is listed in FIG. 5B.

Due to a sequencing error, the nucleotide sequence was initiallybelieved to comprise an additional cytosine residue located between thecytosine residue at position 598 and the adenine residue a position 599,as indicated in the sequence listed in FIG. 1A (SEQ ID NO: 1). Theputative amino acid sequence (SEQ ID NO: 2) encoded by the erroneoussequence is listed in FIG. 1A, and differs from the amino acid sequenceof MCT-1 at amino acid residues 115 to 181.

The dbEST database was compared with the sequence of the MCT-1 cDNA, andseveral overlapping ESTs having homology therewith were identified.These overlapping regions are indicated in FIG. 2. MCT-1 was localizedto the long arm of chromosome Xq22-24 using bacterial artificialchromosome (BAC) clones which contained either the 5′-region, the3′-region, or both regions of the MCT-1 cDNA.

Although there was no significant homology between MCT-1 and any knownprotein at the primary sequence level, there was one interestingalignment at the structural protein level, as assessed using the NRL-3DDatabase (Amino Acid Sequence Extraction, Brookhaven StructuralDatabase). A potentially important region (SEQ ID NO: 9) of MCT-1 is theamino terminal half of the protein, which has a sequence identity of 32%with a 58-amino-acid-residue domain (SEQ ID NO: 10) of cyclin H, asindicated in FIG. 4. It is understood that proteins of theimmunoglobulin superfamily have only, on average, about 23% sequenceidentity. It is known from crystallographic studies that the basic threedimensional folding patterns among this superfamily of proteins isextremely well conserved.

The region of homology between MCT-1 and cyclin H covers a region ofcyclin H that spans a surface domain of the protein that is putativelyinvolved in protein-protein interactions (Andersen et al., 1997, EMBO J.16:958-967). The non-homologous regions of MCT-1 and cyclin H correspondto regions of random coil within the cyclin H molecule. Therefore, MCT-1may be predicted to exhibit high structural homology with cyclin H.Thus, the homologous region of MCT-1 may also be involved inprotein-protein interactions, and MCT-1 was hypothesized to have a rolein cell cycle regulation.

In order to test this hypothesis, MCT-1 was overexpressed in NIH 3T3fibroblasts, and the effect on growth of those cells was observed.Following transfection of the fibroblasts, an approximately two-folddecrease in the duration of the G1/S phase of the cell cycle wasobserved, relative to non-transfected fibroblasts. Over expression ofMCT-1 increases the proliferative rate of cells by decreasing the lengthof G1 phase without causing a reciprocal increase in the durations ofthe S or G2-M phases.

The transforming ability of MCT-1 was assessed by soft agar growthassays. Soft agar culture assays were performed essentially as described(Hsiao et al., 1989, Mol. Cell Biol. 9:2641-2647). Using limitingdilution, clonal cell lines of pcDNA3 and pCMV-MCT-1-transfected cellswere established. Briefly, NIH 3T3 fibroblast monolayers transfectedwith pcDNA3 or pCMV-MCT-1 were seeded into 0.3% (w/v) Bacto-Agarsuspension supplemented with DMEM and 20% (v/v) fetal calf serum. Thissuspension was overlaid above a layer of 0.5% (w/v) agar in the samemedium on a 90 millimeter diameter plate. Samples were made intriplicate and re-fed every four days. Colonies were scored both bynaked eye and by microscopy. Neither the parent cell line nor cellstransfected with pCMV proliferated. Growth of pCMV-MCT-1-transfectedcells was observed after four weeks. Thus, it was demonstrated that onlyMCT-1-overexpressing cells remained viable and continued to proliferate

Some cell cycle regulatory proteins are directly involved inoncogenesis, and cyclins have specifically been implicated intumorigenesis. Amplification of cyclin E has been demonstrated in bothbreast and colon cancer cell lines (Keyomarsi et al., 1993, Proc. Natl.Acad. Sci. USA 90:1112-1116; Leach et al., 1993, Cancer Res.53:1986-1989). The strongest evidence to date for participation ofcyclins in oncogenesis it that cyclin D1 amplification andoverexpression occurs in primary human breast tumors (Buckley et al.,1993, Oncogene 8:2127-2133) and that cyclin D1 overexpression leads totransformation in vitro (Jiang et al., 1993, Oncogene 8:3447-3457).Furthermore, a D-type cyclin, CCND2, has been implicated in cell cycleprogression, and has recently been demonstrated to be amplified innon-Hodgkin's lymphoma (Sherr, 1993, Cell 73:1059-1065; Hoglund et al.,1996, Blood 87:324-330).

The HUT 78 cell line in which MCT-1 amplification was observed wasderived from peripheral blood cells obtained from a patient afflictedwith Sezary syndrome. Because MCT-1 was localized to chromosomal bandsXq22-24, primary samples from patients afflicted with either CTCL (n=40)or chronic lymphocytic leukemia (n=20). Amplification of MCT-1 was notdetected in these samples. Nonetheless, it appears that MCT-1overexpression contributes to deregulated cell cycle progression andproliferation in vitro. Further support for the hypothesis that MCT-1 isan oncogene is provided by the observation that the gene supports softagar growth in fibroblasts which over express it and by the observedstructural homology between MCT-1 and cyclin H.

The materials and methods used in the experiments presented in thisExample are now described.

Chromosomal localization of MCT-1 was ascertained by fluorescent in situhybridization (FISH) analysis. Briefly, a BAC library was screened usingPCR primers homologous with or complementary to the 5′- and 3′-ends ofthe cDNA listed herein in FIG. 1A (SEQ ID NO: 1). Two BAC clones (BAC5839 and BAC 5841) hybridized with both the 5′- and the 3′-primer. EachBAC probe was labeled with dioxigenin dUTP by nick translation. Thelabeled probe was hybridized to normal metaphase chromosomes fromphytohemagglutinin-stimulated peripheral blood lymphocytes. Specifichybridization signals were observed on the long arm of the X chromosome(Xq22-24) using BAC 5839.

Stable cell lines which overexpressed human MCT-1 were established bytransfecting NIH 3T3 fibroblasts either with vector pcDNA3 (comprisingpromoter pCMV) alone or with vector comprising the full length MCT-1cDNA (pCMV-MCT-1). Geneticin (G418) resistance was conferred bypCMV-MCT-1. pCMV-MCT-1 was made using specific restriction enzymes. Togenerate pCMV-MCT-1, the vector comprised the T7 promoter operablylinked to the coding sequence of MCT-1 was generated using primershaving nucleotide sequences (+) 5′-GCTGAGGATC CGGTTGCCTA AAAG-3′ (SEQ IDNO: 5) and (−) 5′-TCTGGTGAAT TCATTCAGCA TAA-3′ (SEQ ID NO: 6), digestedwith BamHI and EcoRI, and ligated to pCMV. These constructs wereverified by DNA sequence analysis.

Transfected cells were grown in selection medium for 2 weeks. Selectionmedium comprised Dulbecco's modified Eagle's medium (DMEM) complete plus1 milligram per milliliter G418. In this medium, 100% of mocktransfected cells exhibited cell death. Controls included cellstransfected with vector (pcDNA3) alone and non-transfected 3T3.Population doubling times were calculated by counting cells every 72hours for 12 days.

After serum depletion for 48 hours, cells were re-plated at 3×10⁵ cellsper 10-centimeter diameter dish in DMEM plus 10% (v/v) fetal calf serum(FCS). Every two hours cells were collected and analyzed for DNA contentby flow cytometry. Fluorescence data was collected using an EpicsCoulter flow cytometer, and the percentage of cells in each of the G1,S, and G2-M phases of the cell cycle were determined by analysis withthe software program, MultiCycle (Phoenix).

AP-PCR

Genomic DNA was prepared from all T-cell lines and normal PBL samples.All reactions were carried out in a 25 microliter volume containing 10millimolar Tris-HCl, 200 millimolar each dNTP, 50 millimolar KCl, 5millimolar MgCl₂, 25 picomoles of 10-mer arbitrary primer, 0.1 milligramDNA template and 1 Unit Taq DNA Polymerase (Fisher Biotech) at pH 8.3. Apanel of 10-mer to 20-mer primers were labeled using T4 polynucleotidekinase and [γ-³²P]-ATP. All reactions were performed using a GeneAMP PCRSystem 9600 (Perkin-Elmer). The profile was as follows. The first 5cycles of the temperature profile:

-   -   denaturation at 95° C. for 30 seconds, then    -   primer annealing at 25° C. for 1 minute, and then    -   extension for 1 minute at 72° C.        The last 25 cycles:    -   denaturation at 95° C. for 30 seconds, then    -   primer annealing at 30° C. for 30 seconds, and then    -   extension for 1 minute at 72° C.        PCR products were separated by electrophoresis in denaturing 8        molar urea/polyacrylamide gels followed by autoradiography.

Cloning and Sequencing of Genomic MCT-1 Sequences Amplified by AP-PCR

The band that appeared to be amplified in the HUT 78 lane relative tothe corresponding bands from other cell lines and normal lymphocytes wasisolated for cloning and sequencing. This band was excised from gels andincubated in 10 milliliters of 1×Assay Buffer A (Fisher Biotech catalogno. FB6000-10) at 90° C. for 10 minutes. Five microliter of eluted DNAwas re-amplified using the same AP-PCR primer as before with MgCl₂concentration of 5 millimolar for 30 cycles at 30° C. The PCR productwas analyzed by electrophoresis in a polyacrylamide gel to confirm itssize and purity. Amplified DNA was cloned into a compatible thymidinepMOSBlue T-vector (Amersham, Arlington Heights, Ill.). The presence ofan appropriate insert was determined using direct colony PCR using T7and U19-mer pMOSBlue specific primers. Sequencing was performed using anestablished methodology. Sequences obtained from several clones wascompared to known sequences in the GenBank data base using the BLASTnand BLASTx computer programs (Altschul et al., 1990, J. Mol. Biol.215:403-410).

Isolation and Sequencing of MCT-1 cDNA

Full length MCT-1 cDNA sequence was obtained by the RACE method aspreviously described using normal peripheral blood lymphocyte (PBL) cDNA(Frohman, 1993, Meth. Enzymol. 218:340-356).

Southern Blot Analysis

5 To 10 micrograms of genomic DNA of cells obtained from humansafflicted with CTCL or CLL was digested with either HindIII or EcoRI andelectrophoresed in 1.0% (w/v) agarose gels. Transfer to nitrocellulosemembrane and subsequent hybridization was performed using standardmethods. An MCT-1 cDNA probe was random primed with PRIME-IT kit(Stratagene, La Jolla, Calif.) and purified using PrimeErase Quikcolumns (Stratagene) according to the supplier's directions. Theoccurrence of gene amplification was assessed by comparing the ratio ofMCT-1 to β-actin signals. Quantification was carried out using a STORMphosphorimager 860 (Molecular Dynamics, Sunnyvale, Calif.).

EXAMPLE 2 Increased G1 Cyclin/cdk Activity in Cells Overexpressing MCT-1

The materials and methods used in the experiments presented in thisExample are now described.

NIH 3T3 Cell Culture and DNA Transfection

Stably transfected NIH 3T3 cell lines which constitutively expressedMCT-1 (i.e. which comprised plasmid pCMV-MCT-1) and stably transfectedcell lines which comprised a control (pCMV) vector were generated asdescribed in Example 1. Individual clones of transfected cells wereobtained by limiting dilution.

Transient assays of the level of MCT-1 protein expression were performedusing the Lipofectamine method according to the supplier's instructions(GIBCO, Grand Island, N.Y.). Briefly, an expression vector(pcDNA-HA-MCT-1, which encodes a protein having an HA tag fused at theamino terminus of MCT-1) was constructed by cloning cDNA encoding MCT-1into BamHI and EcoRI site of pcDNA3-HA. Transfected cells were analyzed48 hours later for production of the fusion protein HA-MCT-1 using ananti-HA antibody described herein.

Lymphocyte Cell Lines

PBL (peripheral blood lymphocytes) were prepared from whole fresh bloodof healthy donors. Mononuclear cells were isolated by centrifugation inthe presence of Ficoll (Organon Teknika Corporation, Durham, N.C.),cultured for 48 hours in RPMI 1640 medium containing 20% (v/v) FCS, 100Units per milliliter penicillin, 100 micrograms per milliliterstreptomycin, 2 millimolar L-glutamine, and 1% (w/v) phytohemagglutinin(PHA; GIBCO, Grand Island, N.Y.). Non-adherent PBL were viably frozenfor further analysis.

Interleukin-2- (IL-2-)independent cell lines which were used includedcell lines C10MJ, MT-2, Hut 78, H-9, HUT 102, DA 202, and C91PL(Advanced Biotechnologies Inc., Columbia, Md.). Cells of these lineswere cultured in RPMI 1640 medium containing 10% FCS, 100 Units permilliliter penicillin, 100 micrograms per milliliter streptomycin and 2millimolar glutamine (GIBCO, Grand Island, N.Y.). IL-2-dependent celllines N1185 and N1186 (described by Berneman et al., 1992, Proc. Natl.Acad. Sci. USA 89:3005-3009) were cultured using the same cultureconditions as above with the addition to the medium of 40 Units permilliliter of recombinant IL-2 (GIBCO, Grand Island, N.Y.).

Immunoprecipitation and Immunoblotting

Cell pellets were lysed in lysate buffer, which comprised 10 millimolarTris, 150 millimolar NaCl, 1 millimolar EDTA, 0.1% (w/v) SDS, and 150millimolar phenylmethylsulfonyl fluoride (PMSF) at a pH of 7.4. Totalprotein concentration in each sample was determined using a commercialbicinchoninic acid assay kit (micro-BCA™ kit; Pierce, Rockford, Ill.)according to the manufacturer's instructions. Equal amounts of wholecell lysate (50 to 100 micrograms) were resuspended in 5 milliliters ofTB S containing (final concentrations) 1 microgram per milliliterleupeptin, 1 microgram per milliliter aprotinin, 0.01% (w/v) PMSF, 0.01%(w/v) n-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), 0.01% (w/v)n-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), 0.1% (w/v) sodiumazide and 1% (w/v) nonyl phenoxy polyethoxy ethanol (NP-40; SIGMA, SaintLouis, Mo.). TBS was Tris-buffered saline containing 0.1% (w/v) sodiumdodecyl sulfate. Samples were pre-cleared with Protein-G beads (GIBCO,Grand Island, N.Y.) and either normal rabbit or mouse serum (each at a1:1000 dilution). Immunoprecipitation of cyclin D1 was carried out for12 hours at 4° C. Immune complexes were precipitated using 1 to 5micrograms of antibody and Protein-G agarose, and were then heated at95° C. for 5 minutes in IP buffer, which comprised 93 millimolar Tris,3% (w/v) SDS, 1.1 millimolar β-mercaptoethanol, 0.03% (w/v) bromophenolblue (BPB), and 15% (v/v) glycerol (SIGMA, Saint Louis, Mo.) at a pH of6.8. The eluant was analyzed using a denaturing, reducing SDS-PAGE gel,and the contents of the gel were transferred to supported nitrocellulosefilter by electroblotting. Filters were incubated with 1 to 5 microgramsof an antibody which binds specifically with one of the following:cyclin D1, cdk4, cdk6, PCNA, and p21. Horseradish peroxidase-linkedanti-mouse or anti-rabbit whole antibody was used as a secondaryantibody. Chemiluminescence was detected using an ECL™ kit (AmershamLife Science, Arlington Heights, Ill.) according to the manufacturer'sinstruction.

Immune Complex Protein Kinase (CDK4 AND CDK6) Assays

NIH 3T3 cell pellets were lysed in lysate buffer. Total proteinconcentration in each sample was determined using the micro BCA™ kit(Pierce, Rockford, Ill.) according to the manufacturer's instructions.50 Microgram aliquots of cell extract were transferred to individualmicrofuge tubes, and the total volume was brought to 500 microliterswith IP buffer. Immunoprecipitation was performed by incubating thesesolutions overnight at 4° C. in the presence of 2.5 micrograms of mousemonoclonal anti-cdk4 antibody or anti-cdk6 antibody, followed byincubation for 4 hours in the presence of 25 microliters of proteinG-agarose beads. This amount of beads provided an excess of G-agarose,relative to antibody. Precipitated protein pellets were washed 3 timesusing ice-cold lysate buffer and then resuspended in 20 milliliters ofice-cold kinase buffer, which comprised 50 millimolar HEPES buffer, 80millimolar β-glycerophosphate, 2.5 millimolar ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 millimolarMgCl₂, 1 millimolar dithiothreitol (DTT), 2.5 millimolar PMSF, 60 KIUper milliliter aprotinin, 10 milligrams per milliliter leupeptin, and 10millimolar cyclin AMP-dependent protein kinase-inhibitory peptide(SIGMA, Saint Louis, Mo.) at a pH of 7.5. 12 Milliliters of a reactionmix which comprised 10 microcuries of γ[³²P]ATP (about 3,000 Curies permillimole; Amersham, Arlington Heights, Ill.), 25 millimolar non-labeledATP, and 200 nanograms of Rb protein as substrate were added to eachsample, and the mixed samples were incubated at 30° C. for 15 minutes.GST-RB was expressed and purified as previously described (Meyerson andHarlow, 1994) as a source of Rb protein for the reaction mix. Kinasereactions were stopped by adding a volume of 2×SDS sample buffer equalto the volume of the mixed sample and boiling this diluted sample for 5minutes. 2×SDS sample buffer comprised 4% (w/v) SDS, 150 millimolar Trischloride, 20% (v/v) glycerol, 0.02% (w/v) BPB, and 2 millimolar sodiumvanadate at a pH of 6.8. Proteins in the boiled, diluted sample wereseparated by SDS-PAGE in a 10% (w/v) gel. After drying, the gel wasassayed by autoradiography to detect labeled proteins.

Antibodies

Monoclonal and polyclonal antibodies designated HD11 (anti-cyclin D1),PC10 (anti-PCNA), F-5 (anti-p21), H-303 (anti-cdk4), and H-230(anti-cdk6) were obtained from Santa Cruz Biotechnology (Santa Cruz,Calif.). Polyclonal MCT-1 antibodies were generated by inoculatingrabbits with a synthetic peptide corresponding to the first 20 aminoacids at the amino terminus of MCT-1. Immune sera was provided byResearch Genetics (Huntsville, Ala.).

Western Blot

Cultured cells (5×10⁶ to 10×10⁶) were washed three times with phosphatebuffered saline (PBS). Cells were collected by centrifugation, and thepellet was lysed in lysate buffer. The total protein concentration ineach sample was determined using the micro BCA™ kit (Pierce, Rockford,Ill.) according to the manufacturer's instructions. 25 Micrograms oftotal protein per sample was fractionated by electrophoresis in aTris-glycine PAGE gel (Novex, San Diego, Calif.) under denaturing,reducing conditions. The proteins in the gel were transferred tosupported nitrocellulose filters using an electroblotting apparatus(Millipore, Marlborough, Mass.). Replicate filters were incubated eitherwith anti-cyclin D1 antibody or with MCT-1 immune sera.Chemiluminescence was performed using an ECL™ kit (Amersham LifeScience, Arlington Heights, Ill.) according to the manufacturer'sinstruction. The filters were then exposed to x-ray film and labeledproteins were quantitated by laser densitometry using a personaldensitometer (Molecular Dynamics, Sunnyvale, Calif.).

Focus Forming Assay

Stable transfectants and cells of the parent cell lines were grown tonear confluence, and were then plated in 100-millimeter tissue culturedishes at a density of about 0.5×10⁶ cells per dish. The cultures werere-fed every 5 to 6 days, and the number of transformed foci wasdetermined after 2 weeks. Focus formation and morphologic changes werevisualized both by the naked eye and by microscopy after incubation for1 hour with Coomassie blue. All experiments were reproduced at leastthree times for each DNA transfected.

The results of the experiments presented in this Example are nowdescribed. These experiments were designed to examine the impact ofMCT-1 overexpression on protein kinase-mediated G1 phase checkpoints.The kinase activity of two cyclin D1-associated cdks, cdk4 and cdk6, wasexamined in NIH 3T3 cells which constitutively expressed MCT-1. Thecellular levels of cyclin D1 protein, and the association of cyclin D1with cdk4, cdk6, PCNA, and p21 were also assessed.

Steady state cellular protein levels of MCT-1 in NIH 3T3 cells stablytransfected with pCMV-MCT-1 or with control vector (pCMV), as well asnon-transfected 3T3 cells, were assessed by Western blot analysis. Anapproximately 20 kilodalton band was detected with a greater intensityin cell lysates obtained from cells transfected with pCMV-MCT-1 than inlysates obtained from non- or control-transfected cells. Similar resultswere obtained using a transient transfection assay involving HA-taggedMCT-1 protein. HA-tagged MCT-1 protein could be immunoprecipitated fromlysates of cells transfected with pCMV-HA-MCT-1, and HA-tagged MCT-1 wasdetected by Western blotting at a position corresponding to a size ofabout 20 kilodaltons.

An increased level of cyclin D1 protein was detected byimmunoprecipitation in lysates from NIH 3T3 cells transfected withPCMV-MCT-1, relative to asynchronously grown control cells which werenot transfected or which were transfected with control vector. Becausecdk4 and cdk6 associate with cyclin D1 during cell cycle progression,complex formation among these molecules was investigated usingco-immunoprecipitation analysis. Increased subunit complex formation inNIH 3T3 cells constitutively expressing MCT-1, relative to cells whichwere not transfected with a vector encoding MCT-1. Physical interactionof PCNA with cyclin D1-cdk4/cdk6 complexes was also investigated.Co-immunoprecipitation of cyclin D1 and PCNA was detected at anincreased level, relative to cells which were not transfected with avector encoding MCT-1. Direct physical interaction was not detectedbetween MCT-1 and any of proteins cyclin D1, cdk4, cdk6, and PCNA underthese assay conditions.

Previous studies demonstrated that ectopic expression of cyclin D1 caninduce transcriptional activation of the p21 gene (Hiyama et al., 1997,Oncogene 14: 2533-2542). In normal human fibroblasts, the cdk inhibitoryprotein p21 can be detected in association with various cyclin/cdkcomplexes in combination with PCNA (Zhang et al., 1993, Mol. Biol. Cell4:897-906). Therefore, these G1 cyclin/cdk complexes were examined forthe presence p21. Protein p21 was determined to be associated with thesecomplexes in cell lines which constitutively expressed MCT-1.

Previous studies establish that p21 can act as a universal inhibitor ofcyclin/cdk kinase activity (Xiong et al., 1993, Nature 366:701-704).Because increased G1 cyclin/cdk complexes were observed in cells whichoverexpressed MCT-1, as described in this Example, we analyzed thecatalytic activity of cdk4 and cdk6 in extracts made from these cellswere assessed. A markedly increased ability to phosphorylate Rb protein(a substrate of both cdk4 and cdk6) was observed in in vitro immunecomplex kinase assays performed using either cdk4 or cdk6immunoprecipitated from MCT-1-overexpressing cells. Without wishing tobe bound by any particular theory of operation, these data suggest thatassociation of a single p21 molecule with a cyclin/cdk complex permitstables complex formation among cyclin D1, cdk and PCNA.

Using a focus forming assay, the ability of cells overexpressing MCT-1to form foci comprising smaller cells that grew in clusters wasdemonstrated. Focus formation could not be detected among non- orcontrol-transfected cells. These results are consistent with earlierwork showing that fibroblasts overexpressing cyclin D1 exhibitmorphological changes and grow in clusters (Jiang et al., 1993, Oncogene8:3447-3457; Wang et al., 1994, Nature 369:669-671).

MCT-1 and cyclin D1 protein levels were assessed in asynchronously-grownT-cell tumor cells and PBL control cells. A number of the T-cell tumorcell lines exhibited elevated MCT-1 protein levels relative to PBLcontrols. Increased MCT-1 protein expression correlated with increasedlevels of cyclin D1. The two IL-2 dependent cell lines N1185 and N1186exhibited increased levels of cyclin D1, but contained no detectableMCT-1 protein. The HUT 78 cell line had the highest level of MCT-1protein. This result is consistent with gene amplification as describedin Example 1 herein. None of the other tumor cell lines analyzed in thisstudy exhibited MCT-1 gene amplification.

A striking finding of the experiments described in this Example is thestrong correlation between MCT-1 overexpression and elevated cyclin D1protein levels, both in transfected NIH 3T3 cells and in a panel ofT-cell tumor cell lines. These experiments demonstrate for the firsttime that MCT-1 protein is endogenously expressed in tumor cells.Furthermore, these experiments also highlight the biologicalsignificance of genomic amplification of MCT-1 in the HUT 78 cell line,since the level of MCT-1 protein is greatly increased relative to theother cell lines which do not exhibit genomic amplification.

Without wishing to be bound by any particular theory of operation, it isrecognized that these data are consistent with MCT-1 acting through anupstream mechanism(s) involving cyclin D1 resulting in dys-regulation ofG1-associated cdk activity. MCT-1 overexpression induces cells to passthrough cell cycle phase G1. It has been demonstrated by others thatwhen cyclin D1 levels are elevated, as they are in cells constitutivelyexpressing MCT-1 (as described herein), expression of several genesinvolved in growth regulation are induced (Jiang et al., 1993, Oncogene8:3447-3457). Furthermore, the amino terminus of MCT-1 shares a regionof homology with the carboxyl terminal region of cyclin H. As describedin Example 1, this region of cyclin H is known to be involved inprotein-protein interactions (Andersen et al., 1997, EMBO J.16:958-967). Cyclin H forms a ternary complex with proteins cdk7 and MAT1, and together these proteins form the cdk-activating kinase (CAK). TheCAK is responsible for activating cdk1, cdk2 and cdk4 (Nigg, 1996, Curr.Opin. Cell Biol. 8:312-317).

Thus, still not wishing to be bound by any particular theory ofoperation, a plausible explanation for the rapid progression of MCT-1overexpressing cells through the G1 phase is enhancement of CAK activityand increased cyclin D1 protein expression, which is coupled withenhanced expression of other growth regulating genes. The experimentsdescribed in this Example demonstrate that overexpression of MCT-1results in loss of normal cell cycle regulatory controls with anincrease in G1 cyclin/cdk complex formation. While the underlyingmechanisms are not known at present, dys-regulation of MCT-1 appears tobe a potent transforming event in vitro, and overexpression is increasedin T-cell tumor cell lines relative to normal lymphocytes. Theseobservations demonstrate that abnormally high levels of expression ofMCT-1 protein can be used as an indicator of the tumor state of a cell,and that tumorigenesis may be inhibited by inhibiting expression ofMCT-1, such as by providing an antisense oligonucleotide which iscomplementary to or homologous with a portion of the gene encoding MCT-1to a cell.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of determining whether a cell expresses MCT-1 proteincomprising the sequence of SEQ ID NO:8, the method comprising:contacting the cell with a composition comprising isolated polyclonalantibodies which bind with specificity to the MCT-1 protein anddetecting binding of the polyclonal antibodies to the protein, whereinthe detecting the binding of the polyclonal antibodies is indicativethat the cell expresses the MCT-1 protein.
 2. The method of claim 1,wherein the cell exhibits deregulated cell cycle progression.
 3. Themethod of claim 2, wherein the cell is a cancer cell.